a) Field of the Invention
The invention is related to a method and an arrangement for optical examination for processing of a sample.
b) Description of the Related Art
At the present time, nonlinear contrasts such as multiphoton absorption or second harmonic generation (SHG) are used increasingly in microscopy, e.g., for examining biological specimens. Short-pulse lasers are advantageously used in order to provide the energy necessary for exciting nonlinear effects. For this purpose, the pulse peak output should be as high as possible and, therefore, the pulse length at the location of the sample should be as small as possible in order to prevent the specimen from being damaged at the same time. Short-pulse lasers deliver light pulses, for example, with a pulse length of some tens of femtoseconds at a repetition rate of some tens of megahertz. Accordingly, they have the advantage of emitting extremely high pulse peak energies with low average output at the same time.
It is disadvantageous that the short pulses change over the path through the microscope to the sample due to group velocity dispersion (GVD); normally, they become longer.
Corresponding arrangements have been proposed to compensate pulse lengthening (DE 19827139A1, DE19744302A1, DE19930532A1).
The described devices are suitable substantially only to compensate second-order dispersion. Therefore, they are not adequate in case higher-order dispersions occur, i.e., pulse lengthening cannot then be compensated completely. However, higher-order dispersions that cannot be determined beforehand must be taken into account, for example, in biological specimens. Further, higher-order dispersions occur in the optical components in a microscope. Accordingly, it is not possible to create optimal conditions for exciting nonlinear contrasts using the conventional techniques.
Another disadvantage consists in that the samples can be damaged by the high pulse peak outputs or pulse peak intensities outside the area in which sample interaction is desired, or the optics of the microscope arrangement can be damaged.
In conventional fluorescence microscopy, different dyes are used for specific marking of biological specimens. These dyes are subsequently excited by different light wavelengths. In specimens of this kind, a simultaneous excitation of the different dyes is usually carried out through the use of multiphoton excitation. This is advantageous on the one hand, since only one light wavelength is needed for excitation. On the other hand, it is disadvantageous when the emission wavelength bands of the individual dyes overlap because the dyes can then no longer be separated spectrally.